Nuclear Ndp52 - a Putative Transcription Regulator

Abstract

NDP52/CALCOCO2 has been well characterised as an autophagy receptor, however a population of the protein exists in the nucleus. Moreover, NDP52 shares significant homology to a transcription co-activator CoCoA. We have recently described the activation of Myosin VI by NDP52 in RNA Polymerase II (RNAPII)-dependent transcription. Although depletion of NDP52 is known to reduce the steady-state levels of mRNA in cells, the molecular role of NDP52 in the nucleus is not clear. We are using cell biology combined with biophysical techniques to understand the functions of NDP52 in nucleus. Using SEC-MALS, Microscale Thermophoresis and Electron Microscopy, we can show that NDP52 is an elongated dimer of approximately 110 kDa that is capable of binding double-stranded DNA in vitro with high affinity. This interaction can also be observed in cells through Chromatin Immunoprecipitation (ChIP). We observed NDP52 bound to promoter regions of several oestrogen-responsive genes. When we knock-down NDP52 in these cells, we see dysregulation of known gene-targets of the oestrogen receptor, as well as of those relating to apoptosis, DNA repair and recruitment of RNAPII. Our proteomics data now suggest that this regulation might not only be sustained through direct interactions with DNA, but also through protein-protein interactions with binding partners of NDP52 in the nucleus. Overall, we propose that NDP52 may be functioning as a transcription regulator in human cells. To test this idea, we have started using super-resolution imaging - PALM-STORM, combined with Cluster analysis software, to image NDP52 at single-molecule level in combination with RNAPII and other candidate binding partners.